Methods, Devices, And Compositions For Treating Vascular Aneurysms

ABSTRACT

Methods, compositions, and devices for treating a vascular aneurysm, including an abdominal aortic aneurysm, are disclosed. In particular, the various embodiments relate to a method of treating an abdominal aortic aneurysm by increasing the mechanical stiffness of an aortic segment adjacent to the abdominal aortic aneurysm in a subject. The mechanical stiffness of the adjacent aortic segment may be increased, for example, by applying a surgical adhesive or intravascular stent. Such treatment reduces stress on the aortic wall and limits further growth of the abdominal aortic aneurysm.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims priority as a continuation to U.S. patentapplication Ser. No. 15/070,817, filed Mar. 15, 2016, and entitled“Methods, Devices, and Compositions for Treating Abdominal AorticAneurysms,” which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application 62/133,450, filed Mar. 15, 2015, and entitled“Methods of Treating Abdominal Aortic Aneurysms,” which is herebyincorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with Government support under contract1R01HL105299 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The various embodiments herein relate generally to methods of treatingan abdominal aortic aneurysm (also referred to herein as an “AAA”). Inparticular, the implementations relate to methods, devices, andcompositions for treating AAA disease by increasing the mechanicalstiffness of an aortic segment adjacent to an abdominal aortic aneurysmin a subject.

BACKGROUND OF THE INVENTION

Abdominal aortic aneurysm carries a high mortality in case of rupture.Current therapies are limited to open surgical or interventionalstent-based exclusion of the aneurysmal sac from the circulation inorder to prevent rupture. However, these treatment options are generallyreserved for larger aneurysms (typically AAA diameter greater than 5.5cm), and there is no effective therapy targeting the evolution of smallaneurysms. The lack of treatment options partly derives from aninsufficient understanding of early AAA pathogenesis.

Recent evidence suggests that AAA formation is not simply due to aorticwall degeneration, resulting in passive lumen dilation, but to active,dynamic remodeling. The latter involves transmural inflammation,extracellular matrix (“ECM”) alterations including elastin fragmentationand (compensatory) collagen deposition, vascular smooth muscle cell(VSMC) apoptosis, and oxidative stress.

From a patho-mechanistic point of view, it is essential not only tocharacterize the particular cellular and molecular alterations involvedin AAA formation, but also to identify early triggers of remodeling.

There remains a need for better methods of treating AAA, particularly atearly stages of disease.

BRIEF SUMMARY OF THE INVENTION

Discussed herein are various methods of treating an abdominal aorticaneurysm. Other embodiments relate to methods of treating any type ofvascular aneurysm, including, for example, thoracic aortic aneurysms.

In one aspect, the invention includes a method of treating a subject foran abdominal aortic aneurysm, the method comprising increasing themechanical stiffness of an aortic segment adjacent to the abdominalaortic aneurysm in the subject. In one embodiment, increasing themechanical stiffness of the aortic segment comprises applying a surgicaladhesive locally to the aortic segment. In another embodiment,increasing the mechanical stiffness of the aortic segment comprisesdeploying an intravascular stent that stiffens the aortic segment. Thesubject may have early stage, intermediate stage, or late stage AAAdisease, wherein the treatment reduces the growth of an abdominal aorticaneurysm compared to in the absence of the treatment. The subject mayfurther show decreased inflammation, apoptosis, or reactive oxygenspecies in the abdominal aorta as a result of the treatment.

In another aspect, the invention includes a method of minimizing growthof an abdominal aortic aneurysm in a subject, the method comprisingincreasing the mechanical stiffness of an aortic segment adjacent to theabdominal aortic aneurysm.

More specifically, in Example 1, a method of treating a subject for anabdominal aortic aneurysm comprises increasing the mechanical stiffnessof an aortic segment adjacent to the abdominal aortic aneurysm in thesubject.

Example 2 relates to the method according to Example 1, whereinincreasing the mechanical stiffness of the aortic segment comprisesapplying a surgical adhesive locally to the aortic segment.

Example 3 relates to the method according to Example 1, whereinincreasing the mechanical stiffness of the aortic segment comprisesdeploying an intravascular stent that stiffens the aortic segment.

Example 4 relates to the method according to Example 1, wherein growthof the abdominal aortic aneurysm is reduced compared to in the absenceof treating the subject.

Example 5 relates to the method according to Example 1, wherein thesubject shows decreased inflammation in the abdominal aorta compared toin the absence of treating the subject.

Example 6 relates to the method according to Example 1, wherein thesubject shows decreased apoptosis in the abdominal aorta compared to inthe absence of treating the subject.

Example 7 relates to the method according to Example 1, wherein thesubject shows decreased production of reactive oxygen species in theabdominal aorta compared to in the absence of treating the subject.

Example 8 relates to the method according to Example 1, wherein thesubject has an early stage abdominal aortic aneurysm.

Example 9 relates to the method according to Example 1, wherein thediameter of the abdominal aortic aneurysm is less than 5.5 cm.

In Example 10, a method of minimizing growth of an abdominal aorticaneurysm in a subject comprises increasing the mechanical stiffness ofan aortic segment adjacent to the abdominal aortic aneurysm.

Example 11 relates to the method according to Example 10, whereinincreasing the mechanical stiffness of the aortic segment comprisesapplying a surgical adhesive locally to the aortic segment.

Example 12 relates to the method according to Example 10, wherein growthof the abdominal aortic aneurysm is reduced compared to in the absenceof increasing the mechanical stiffness of the aortic segment.

Example 13 relates to the method according to Example 10, wherein thesubject shows decreased inflammation in the abdominal aorta compared toin the absence of increasing the mechanical stiffness of the aorticsegment.

Example 14 relates to the method according to Example 10, wherein thesubject shows decreased apoptosis in the abdominal aorta compared to inthe absence of increasing the mechanical stiffness of the aorticsegment.

Example 15 relates to the method according to Example 10, wherein thesubject shows decreased production of reactive oxygen species in theabdominal aorta compared to in the absence of increasing the mechanicalstiffness of the aortic segment.

Example 16 relates to the method according to Example 10, wherein thesubject has an early stage abdominal aortic aneurysm.

Example 17 relates to the method according to Example 10, wherein thediameter of the abdominal aortic aneurysm is less than 5.5 cm.

In Example 18, a method of treating an abdominal aortic aneurysmcomprises positioning a stiffening device or stiffening composition atat least one aneurysm-adjacent aortic segment, whereby the stiffeningdevice or stiffening composition increases the mechanical stiffness ofthe aneurysm-adjacent aortic segment.

Example 19 relates to the method according to Example 18, wherein thestiffening composition comprises a surgical adhesive, wherein thepositioning the stiffening composition further comprises applying thesurgical adhesive to an outer surface of the aneurysm-adjacent aorticsegment.

Example 20 relates to the method according to Example 18, wherein thestiffening device comprises an intravascular stent, wherein thepositioning the stiffening device further comprises deploying theintravascular stent into a lumen of the aneurysm-adjacent aorticsegment.

These and other embodiments will readily occur to those of skill in theart in view of the disclosure herein. While multiple embodiments aredisclosed, still other embodiments of the present invention will becomeapparent to those skilled in the art from the following detaileddescription, which shows and describes illustrative embodiments of theinvention. As will be realized, the invention is capable ofmodifications in various obvious aspects, all without departing from thespirit and scope of the present invention. Accordingly, the drawings anddetailed description are to be regarded as illustrative in nature andnot restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a front, cutaway view of an intravascular stent positioned inan aorta, according to one embodiment.

FIG. 2 is a front view of a surgical adhesive applied to an outersurface of an aorta, according to another embodiment.

FIG. 3 is a schematic depiction of the walls of a normal (healthy) aortaduring diastole, in which the walls are in an unexpanded state.

FIG. 4 is a schematic depiction of the walls of the normal (health)aorta of FIG. 3 during systole, in which the walls are in an expandedstate.

FIG. 5A is a schematic depiction of the walls of a segmentally stiffaorta during diastole.

FIG. 5B is a schematic depiction of the walls of the segmentally stiffaorta during systole.

FIG. 6A is a line graph depicting temporal development ofcircumferential cyclic strain of PPE-treated and saline-treatedsegments.

FIG. 6B is a line graph depicting diameter development of thePPE-treated and saline-treated segments (% vs. baseline (d0)) over time.

FIG. 6C is a line graph depicting temporal analysis of segmental aorticstiffness (“SAS”) of the PPE-treated or saline-treated segment relativeto the adjacent abdominal aorta.

FIG. 6D is a line graph depicting temporal analysis of thecircumferential cyclic strain of the aorta adjacent to the PPE-treatedsegment.

FIG. 6E is a scatter plot graph depicting the correlation between thesegmental aortic stiffness (“SAS”) at day 7 and the consecutive diameterincrease of the PPE-treated segment in the following 7 days.

FIG. 6F is a set of images depicting representative immunofluorescencestaining for collagen I+III (red) with green autofluorescence of elastinlamellae in the upper two panels and Modified Elastin Verhoeff's VanGieson (“VVG”) staining in the lower two panels.

FIG. 7A is a schematic depiction of a finite elements model (“FEA”)showing axial stress analysis of segmental aortic stiffening in Newtonsper square millimeter (N/mm²) in which the stiffness of the stiff aorticsegment was increased (shear moduli: 500 kPa left vessel, 1100 kPamiddle vessel, 1700 kPa right vessel) to demonstrate the impact ofsegmental stiffness on axial stress generation.

FIG. 7B is a schematic depiction of a finite elements model (“FEA”)showing axial stress analysis of segmental aortic stiffening in N/mm² inwhich the intraluminal pressure was increased (left vessel: 80 mmHg,middle vessel: 130 mmHg, right vessel: 180 mmHg) to visualize theinfluence of blood pressure on axial stresses in a segmentally stiffaorta.

FIG. 7C is a schematic depiction of a finite elements model (“FEA”)showing axial stress analysis of segmental aortic stiffening in N/mm² inwhich a segmentally stiff aorta without external stiffening (left) iscompared to a segmentally stiff aorta that is subjected to externalstiffening of the adjacent compliant segments (simulating gluetreatment; right) to demonstrate axial stress reduction andhomogenization induced by the interventional external stiffening.

FIG. 8A is a bar graph depicting temporal analysis of the Col1a1 andCol3a1 gene expression in the AAA-adjacent aorta compared to the AAA(PPE-treated) segment.

FIG. 8B is a bar graph depicting temporal analysis of miR-29b expressionin the AAA-adjacent aorta compared to the AAA (PPE-treated) segment.

FIG. 8C is a set of images depicting in situ hybridization for miR-29b(purple-blue dye) and red nuclear counterstain in the AAA-adjacentaortic segments (original magnification 400×, scale bar 50 μm) at day 0and day 7.

FIG. 8D is a set of images depicting the aortic wall taken fromAAA-adjacent aortic segments 7 days or 14 days after PPE-treatment, withthe upper panels showing the segments stained with Picrosirius Red (red:collagen; yellow: muscle) and the lower panels showing the segmentsstained with Elastin VVG staining.

FIG. 9A is a line graph depicting temporal analysis of thecircumferential cyclic strain of the glue-treated adjacent aorta (boldline) in relation to the PPE-treated segment (thin line).

FIG. 9B is a line graph depicting temporal analysis of segmental aorticstiffness (“SAS”) in glue-treated aortas compared to sham-glue-treatedconditions.

FIG. 9C is a line graph depicting diameter development of thePPE-treated segment in glue-treated vs. sham-glue-treated conditions.

FIG. 9D is a line graph depicting temporal development of SAS followingdelayed glue or sham treatment 7 days (as identified by the arrow) afterPPE surgery.

FIG. 9E is a line graph depicting temporal development of aorticdiameter following delayed glue or sham treatment 7 days (as identifiedby the arrow) after PPE surgery.

FIG. 9F is a set of images depicting representative Elastin VVG stainingof the aortic wall taken from native abdominal aortas (control) orPPE-treated segments at day 14 after additional treatment of theadjacent aorta with glue or sham-glue (original magnification 400×;scale bars 50 μm).

FIG. 9G is a set of images depicting representative Picrosirius Redstaining of the aortic wall taken from native abdominal aortas (control)or PPE-treated segments at day 14 after additional treatment of theadjacent aorta with glue or sham-glue (original magnification 400×;scale bars 50 μm).

FIG. 10A is a set of images depicting in situ DHE staining of nativeabdominal aortas (control) or PPE-treated segments after additionaltreatment of the adjacent aorta with glue or sham-glue at day 7.

FIG. 10B is a bar graph depicting average fluorescence from three highpower fields of three different aortas per group (control, sham, andglue).

FIG. 100 is a set of images depicting representative co-staining ofmacrophages (red F4/80 marker) and the green labeled cytokine IL-6 innative abdominal aortas (control) or PPE-infused segments at day 7 afteradditional treatment of the adjacent aorta with glue or sham-glue(original magnification 400×, scale bar 50 μm).

FIG. 10D is a set of images depicting representative co-staining ofmacrophages (red F4/80 marker) and the green labeled cytokine IL-1β innative abdominal aortas (control) or PPE-infused segments at day 7 afteradditional treatment of the adjacent aorta with glue or sham-glue(original magnification 400×, scale bar 50 μm).

FIG. 10E is a set of images depicting representative co-staining ofmacrophages (red F4/80 marker) and the green labeled cytokine Ccl2 innative abdominal aortas (control) or PPE-infused segments at day 7 afteradditional treatment of the adjacent aorta with glue or sham-glue(original magnification 400×, scale bar 50 μm).

FIG. 10F is a set of images depicting corresponding immunostaining ofactivated caspase-3 (red).

FIG. 10G is a bar graph depicting expression of II6, Ccl2, and II1b inthe PPE-infused segment at day 7 after additional glue or sham-gluetreatment of the adjacent aorta, quantified in whole tissue.

FIG. 10H is a bar graph depicting expression of II6, Cc/2, and II1b inthe PPE-infused segment at day 7 after additional glue or sham-gluetreatment of the adjacent aorta, quantified in laser-capturedmacrophages.

FIG. 10I is a bar graph depicting expression analysis of Mmp2 and Mmp9in the PPE-infused segment at day 7 after additional glue or sham-gluetreatment of the adjacent aorta.

FIG. 10J is a bar graph depicting expression analysis of Col1a1 andCol3a1 in the PPE-infused segment at day 7 after additional glue orsham-glue treatment of the adjacent aorta.

FIG. 11A-1 depicts an unrestrained/unstiffened aorta upon which cyclicstrain is imposed in Example 1 as described below.

FIG. 11A-2 depicts a completely restrained aorta upon which cyclicstrain is imposed in Example 1 as described below.

FIG. 11A-3 depicts a segmentally restrained aorta upon which cyclicstrain is imposed in Example 1 as described below.

FIG. 11B is a bar graph depicting the differential expression ofinflammation related genes II6 and Ccl2 after one hour of mechanicalstimulation of the three groups of aortas.

FIG. 11C is a bar graph depicting the differential expression of matrixmetalloproteinases Mmp2 and Mmp9 after one hour of mechanicalstimulation of the three groups of aortas.

FIG. 11D is a bar graph depicting the differential expression ofcollagen genes Col1a1 and Col3a1 after one hour of mechanicalstimulation of the three groups of aortas.

FIG. 12A is a scatter plot graph depicting the correlation between ageand circumferential cyclic strain in the supra-renal segment of thehuman abdominal aorta.

FIG. 12B is a scatter plot graph depicting the correlation between ageand circumferential cyclic strain in the mid-infrarenal segment of thehuman abdominal aorta.

FIG. 12C is a scatter plot graph depicting the correlation between ageand circumferential cyclic strain in the bifurcational segment of thehuman abdominal aorta.

FIG. 12D is a scatter plot graph depicting the correlation between ageand segmental stiffness (SAS, bifurcational segment vs. mid-infrarenalsegment) along the infrarenal abdominal aorta.

FIG. 13 depicts the setup for differential mechanical stimulation of themurine aorta ex vivo in Example 1 as described below.

FIG. 14A is a photograph of the abdominal aorta before glue application.

FIG. 14B is a photograph of the abdominal aorta after glue applicationto the segments adjacent to the PPE-treated segment, as described inExample 1 below.

FIG. 15 is a bar graph depicting shear stress occurring in thePPE-treated segment as captured by ultrasound measurements on day 7after PPE-induction.

FIG. 16 is a set of images depicting elastin architecture via VerhoefVan Gieson staining in the upper panels and collagen deposition viaPicrosirius Red staining in the lower panels of the AAA-adjacent aortaon day 14 with glue treatment in the right panels and without gluetreatment in the left panels.

FIG. 17 is a set of images depicting immunofluorescence macrophageco-staining for inflammatory cytokines IL-6, IL-1β and Ccl2 on day 7after PPE-treatment.

FIG. 18A is a photograph of macrophages with F4/80-fluorescent labels.

FIG. 18B is a photograph of macrophages being selectively targeted.

FIG. 18C is a photograph of macrophages being isolated via laser capturemicrodissection (“LCM”).

FIG. 18D is a bar graph depicting confirmation of macrophage transcriptenrichment via enhanced Emr1 expression compared to LCM-isolatedF4/80-negative cells.

FIG. 19 is a flow chart describing the mechanism of early AAA formationdriven by age-related segmental aortic stiffening, according to oneembodiment.

DETAILED DESCRIPTION

The various embodiments disclosed or contemplated herein relate tomethods, systems, and devices for treating an abdominal aortic aneurysm.More specifically, the various implementations involve treating anabdominal aortic aneurysm in a subject by increasing the mechanicalstiffness of an aortic segment adjacent to the aneurysm, therebyreducing the stress to the aortic wall and limiting further growth ofthe aneurysm. The mechanical stiffness of the adjacent aortic segmentmay be increased, in one exemplary embodiment, by applying a surgicaladhesive to the segment. Alternatively, the mechanical stiffness of theadjacent segment can be increased by implanting an intravascular stent.Other embodiments relate to treatment of any type of vascular aneurysm,including, for example, thoracic aortic aneurysms, wherein the treatmentincludes increasing the mechanical stiffness of adjacent vascularsegments according to, or in similar fashion to, the various methods,devices, and compositions disclosed or contemplated herein.

According to one embodiment, FIG. 1 depicts an intravascular stent 10disposed within the abdominal aorta 12. The stent 10 is disposed withinthe lumen 14 of the aorta 12 and adjacent to the aortic aneurysm 18 asshown. In this specific exemplary embodiment, the stent 10 is positionedadjacent to and upstream from the aneurysm 18. Alternatively, the stent10 can be positioned adjacent to and downstream from the aneurysm 18. Ina further alternative, two stents could be used with one positionedadjacent to and upstream from the aneurysm 18 and the other positionedadjacent to and downstream from the aneurysm 18. In certainimplementations, the stent 10 an expandable stent 10 that is configuredto expand into contact with the inner wall 16 of the aorta, therebyproviding support and mechanical stiffness to the length of aorta 12with which the stent 10 is in contact. In one implementation, the stent10 is a deployable stent 10 that is implanted via a known non-invasiveprocedure. Alternatively, the stent 10 can be any known stent forproviding mechanical intravascular support that can be implanted by anyknown procedure.

FIG. 2 depicts a surgical adhesive 30 applied to or disposed on an outerwall 34 of an aorta 32, according to another implementation. In oneembodiment, the adhesive 30 is a surgical adhesive or surgical glue(also referred to herein as “adhesive” or “gel”). In one specificexample, the adhesive 30 is BioGlue™, which can be purchased fromCryoLife, Inc., in Kennesaw, Ga. Alternatively, the adhesive 30 can beany known medical composition that can be applied to human tissue andsubsequently harden. In this specific exemplary embodiment, the adhesive30 is disposed adjacent to and upstream from the aneurysm 36.Alternatively, the adhesive 30 can be positioned adjacent to anddownstream from the aneurysm 36. In a further alternative, the adhesive30 can be positioned in two locations, with adhesive 30 applied adjacentto and upstream from the aneurysm 36 and further applied adjacent to anddownstream from the aneurysm 36 as well. In use, the adhesive 30 isapplied to the outer wall 34 of the aorta 32 and then allowed to harden,thereby providing mechanical stiffness to the length of aorta 32 thatthe adhesive 30 is disposed along. The adhesive 30 can be applied by anyknown procedure for applying an adhesive to an outer wall of a bloodvessel.

As mentioned above, either of the two devices, compositions, or methodsdepicted in FIGS. 1-2 and discussed above can be used to treat anabdominal aortic aneurysm. More specifically, each of the stent 10 andthe adhesive 30 are positioned as described above to increase themechanical stiffness of the aortic segment along which they arepositioned adjacent to the aneurysms 18, 36 respectively. As such, thestent 10 and the adhesive 30 are each positioned along the aorta toreduce the stress to the aortic wall and limit further growth of theaneurysm 18, 36 respectively.

Without being limited by theory, it is believed that each of theseinterventional mechanical stiffening instruments (10, 30, respectively)positioned adjacent to an abdominal aortic aneurysm are effective inlimiting the growth of the aneurysm (18, 36, respectively) by limitingthe remodeling and expansion of the aneurysm and thereby forestalling oreliminating the need for surgical repair.

It is known that abdominal aortic aneurysm (“AAA”) formation isaccompanied by increased stiffness of the aneurysmal vessel segmentcompared to the normal aorta, also called segmental aortic stiffening(“SAS”). Aneurysmal stiffening occurs due to profound changes inextracellular matrix (“ECM”) organization including elastinfragmentation and enhanced adventitial collagen deposition and turnover.It is believed that the segmental aortic stiffening is a pathogeneticfactor contributing to the development of an abdominal aortic aneurysm.That is, degenerative stiffening of the aneurysm-prone aortic wall leadsto axial stress, generated by cyclic tethering of adjacent, morecompliant wall segments. Axial stress then induces and augmentsprocesses necessary for aneurysm growth such as inflammation andvascular wall remodeling, as will be shown in further detail in theexamples below.

FIGS. 3-5B depict the concept of segmental aortic stiffness generatingaxial wall stress during systolic aortic expansion. More specifically,FIGS. 3 and 4 depict a healthy aorta 40 that is homogenously expandable,while FIGS. 5A and 5B depict a segmentally stiff aorta 50. In FIG. 3,the aorta 40 is shown in its unexpanded state during diastole, whileFIG. 4 depicts the aorta 40 in systole when the aorta 40 is in itsexpanded state. Note that the healthy aorta 40 is homogenouslyexpandable due to the natural compliance of the aorta 40—it has nosegmental aortic stiffening. In contrast, the aorta 50 as shown in FIGS.5A and 5B exhibits segmental aortic stiffening, with the aorta 50 havinga stiff segment 52 in which the walls are relatively stiffer than therest of the aorta 50 and a normal segment 54 having normal, compliantwalls. As shown in FIG. 5A, the segmentally stiff aorta 50 in diastolehas an unexpanded state that looks similar to that of a health aorta(such as the aorta 40 discussed above), with both the stiff segment 52and the normal segment 54 having similar diameters. However, as depictedin FIG. 5B, the aorta 50 in systole has an expanded state in which thestiff segment 52 does not expand (or not as much) while the normalsegment 54 does. As a result, the segmentally stiff aorta 50 issubjected to cyclical, axially tethering forces as depicted by arrowsA1, A2 during the repeated circumferential expansion of the compliantwall segments of the normal segment 54 adjacent to the stiff segment 52during systole. These tethering forces cause axial stress, which resultsin inflammation and vascular wall remodeling of the aortic wall, therebyultimately causing the grown of an aneurysm. In other words, theexistence of a stiff aortic segment adjacent to a more compliant aortagenerates axial wall stress due to non-uniform systolic walldeformations, thereby modulating early aneurysm pathobiology.

The interventional mechanical stiffening of an aneurysm-adjacent aorticsegment as disclosed in the various embodiments herein—including thestent, adhesive, and gel embodiments discuss above—limits AAA remodelingand expansion.

Below are examples of specific embodiments relating to theinterventional mechanical stiffening of an aneurysm-adjacent aorticsegment. They are provided for illustrative purposes only, and are notintended to limited the scope of the various embodiments in any way.

EXAMPLE

As discussed above, AAA formation is due at least in part to active,dynamic remodeling. Mechanical wall stress was an intriguing candidatefor being an early trigger for remodeling. That is, biomechanical stress(i.e., shear stress, circumferential or axial wall stress) may driveadaptive arterial remodeling in response to altered hemodynamics, butalso may induce inflammation and ECM remodeling, as well as VSMCapoptosis in vascular disease.

AAA growth is accompanied by increasing wall stress. While wall stressdue to the vessel's expanding geometry significantly contributes toeventual rupture of the “mature” AAA, it might appear that wall stresswould be unrelated to the pathophysiology in early, pre-aneurysmalstages, when aortic size has not yet overtly changed. However, enhancedwall stress may still occur due to early aortic biomechanicalalterations (i.e., aortic stiffening).

A porcine pancreatic elastase (“PPE”) infusion model was created. Morespecifically, the PPE infusion model to induce AAA in 10-week-old maleC57BL/6J mice was performed as described in Azuma J, Asagami T, DalmanR, Tsao P, “Creation of murine experimental abdominal aortic aneurysmswith elastase,” J Vis Exp. 2009; 29:1280. In brief: after placingtemporary ligatures around the proximal and distal aorta, an aortotomywas created at the bifurcation and an insertion catheter was used toperfuse the aorta for 5 minutes with saline containing porcinepancreatic elastase (1.5 U/mL; Sigma Aldrich).

The PPE-adjacent aortic segments were treated with glue. Morespecifically, in order to locally enhance aortic mechanical stiffness, asurgical adhesive (BioGlue, CryoLife, Atlanta) was applied to thesegments adjacent to the PPE-treated aorta directly after completion ofthe PPE-treatment. Complete polymerization of the two-component glue(albumin/glutaraldehyde) occurred within seconds. As shown in FIG. 14B,care was taken to avoid the PPE-treated segment. For sham-treatmentgroups only one component of BioGlue was applied.

Mouse ultrasound studies were performed. More specifically, systolicdiameter (D_(s)) and diastolic diameter (D_(d)) were quantified in thePPE-treated segment as well as in the adjacent untreated segments usingM-Mode ultrasound. Circumferential cyclic strain c was calculated asε=(D_(s)−D_(d))/D_(d)×100%. Segmental aortic stiffness (SAS) was definedas a relative index to quantify the stiffness of the PPE-treated segmentin relation to the adjacent aorta, calculated asSAS=ε_(adjacent aorta)/ε_(PPEsegment). The strain values for adjacentaorta (ε_(adjacent aorta)) represent an average strain calculated fromthe adjacent segments proximal and distal to the PPE-treated segment.For shear stress calculations, blood flow was assessed as previouslydescribed in Hong G, Lee J, Robinson J, Raaz U, Xie L, Huang N, Cooke J,Dai H., “Multifunctional in vivo vascular imaging using near-infrared IIfluorescence,” Nat Med. 2012; 18:1841-6.

Human ultrasound studies were also performed. Nineteen male volunteersof different ages (youngest age: 36, oldest age: 71, mean age: 51.9years) were included in the study. Exclusion criteria werecardiovascular diseases (in particular AAA), diabetes and history ofsmoking. M-mode images tracking the anterior and posterior aortic wallmotion were recorded at predefined locations (suprarenal, mid-infrarenaland proximal to the aortic bifurcation).

Systolic diameter (D_(s)) and diastolic diameter (D_(d)) were quantifiedin the suprarenal, mid-infrarenal and bifurcational segment of theabdominal aorta to calculate cyclic strain and SAS.

Finite element analyses of the mouse aorta were performed using thecommercial finite element software package ABAQUS. The artery wasmodeled as a 2.0 mm long axisymmetric tube with outer diameter D_(a)=0.9mm and arterial wall thickness t=0.075 mm. The intima, media, andadventitia were summarized in a single homogeneous layer modeled usingan isotropic Neo-Hookean strain energy function with a shear modulus of300 kPa. Stiffness of the stiff segment (I=1.0 mm) was modified asindicated.

An RNA quantification was also performed. Total aortic RNA was isolatedand processed for qRT-PCR using standard protocols and methods.

Laser capture microdissection (“LCM”) was performed as previouslydescribed in Sho E, Sho M, Nanjo H, Kawamura K, Masuda H, Dalman R L,“Comparison of celltype-specific vs. transmural aortic gene expressionin experimental aneurysms,” J Vasc Surg. 2005; 41:844-52. F4/80-stainedmacrophages were micro-dissected from frozen aortic cross sections (7μm) using a PALM MicroBeam System (Zeiss). RNA was subsequentlyprocessed for qRT-PCR using the Single Cell-to-CT Kit (Ambion).

Standard protocols for histology, immunofluorescence, in situ DHEstaining, and in situ hybridization were used.

Ex vivo aortic mechanical stimulation was performed. More specifically,abdominal aortae were explanted, cannulated and mounted in the heatedvessel chamber of a pressure arteriograph system (Model 110P, DanishMyotechnology, Copenhagen, Denmark) and stretched to in vivo length. Theaorta was then subjected to an automated pressure protocol, cyclicallyalternating between 80 mmHg and 120 mmHg with a frequency of 4/minutefor one hour. To stiffen/restrain either the complete aorta or just thecentral segment (to simulate segmental stiffening), a silicone cuff(SILASTIC Laboratory Tubing, inner diameter: 0.51 mm; Dow Corning) wasplaced around the aorta as shown in FIG. 9. After conclusion of theexperiment, the aorta was removed from the cannulas and processed forRNA isolation.

With respect to the statistical information and analysis providedherein, data are presented herein as mean±SEM. For comparison of 2groups, a Mann-Whitney test was performed; for multiple groups (≥3groups), comparison was accomplished by a Kruskal-Wallis test withDunn's posttest. Ultrasound data comparing 2 groups/treatments over timewere analyzed by a permutation F-test based on 2-way repeated measuresANOVA. For each treatment assignment, a repeated measures ANOVA wasperformed and a null distribution of the p-value was derived fortreatment effect. The p-value from the permutation test was thenestablished as the percentage of the null p-values less than the p-valuefrom the real data. To compare ultrasound parameters within onetreatment group over time, the Friedman's test was used. For correlationanalysis of animal ultrasound data, the Spearman correlation was used.For correlation analyses of human ultrasound data, the Pearsoncorrelation was used after passing D'Agostino-Pearson omnibus normalitytest. A value of p≤0.05 (two-sided) was considered statisticallysignificant.

All animal protocols were approved by the Administrative Panel onLaboratory Animal Care at Stanford University (labanimals.stanford.edu/)and followed the National Institutes of Health and USDA Guide lines forCare and Use of Animals in Research.

Results

Based on the results, it can be concluded that aortic stiffeningprecedes aneurysmal dilation in experimental AAA.

The temporal relationship between aortic biomechanical alterations andaneurysmal dilation in the porcine pancreatic elastase (PPE)-infusionmodel of murine AAA was investigated. As shown in FIGS. 6A and 6B,circumferential cyclic aortic strain (as a measure of vascularstiffness) and aortic diameter were monitored over time in thePPE-treated segment and saline-perfused controls via M-Mode ultrasound.

With reference to FIG. 6A, while native abdominal aortae exhibited abaseline cyclic strain of about 12%, PPE-infusion rapidly induced asubstantial strain reduction of more than 50% in the treated segment atd1 followed by further declines until d14, after which it remainedstable until d28. In contrast, saline infusion only resulted in a minorstrain reduction in the corresponding segment.

As shown in FIG. 6B, the aortic diameter, however, displayedinsignificant enlargement up to d7 post-PPE and post-saline. ThePPE-treated segment then dilated markedly between d7 and d14. Afterwardsthe aortic diameter remained relatively stable up to d28.

As evidenced by FIG. 6F, investigating possible mechanisms for the rapidstiffening of the PPE-treated segments we found remarkable elastinfragmentation, while pro-fibrotic responses were only moderate.

With respect to FIGS. 6A-6F, data are mean±SEM, and n=5-13 for eachcondition/time point. Further, p values denote differences between PPEand saline groups by permutation F-test for FIGS. 6A-6C, aortic straindifferences in PPE treated animals over time by Friedman's test for FIG.6D, and significance level of Spearman correlation for FIG. 6E.

The results show that segmental aortic stiffening generates axial wallstress in the AAA-prone segment. Having identified rapid earlymechanical stiffening of the aneurysm-prone segment (i.e. reduced cyclicstrain), the role of that stiffening in aneurysm development wasinvestigated. It was hypothesized that segmental aortic stiffening (SAS;defined as enhanced stiffness of the aneurysm-prone segment relative tothe adjacent aorta) would generate adverse wall stress during cyclicdeformation of the aortic wall, eventually resulting in AAA formation.Thus, in silico wall stress analysis employing a finite element modelwas performed.

Using a simplified approach, the infrarenal mouse aorta was modeled as acylindrical tube in a finite elements model (“FEA”)-based axial stressanalysis of segmental aortic stiffening in which the aorta was subjectedto various mechanical conditions and resulting axial (longitudinal)stress (N/mm²) was depicted as shown in FIGS. 7A-7C. More specifically,to examine the effects of segmental stiffening, a pressure of 130 mmHg(approximating systolic blood pressure) was simulated and a segment ofincreasing stiffness (SS) was introduced adjacent to a non-stiff segment(AS). As shown in FIG. 7A, it was found that increasing segmentalstiffness progressively induced axial stress in the stiff segmentextending from the segmental interface.

As hypertension represents a risk factor for AAA, we explored the impactof high blood pressure levels on axial wall stress by pressurizing ourFEA model with a fixed stiffness of the stiff segment up to 180 mmHg. Asshown in FIG. 7B, this simulation revealed that high blood pressureaugmented segmental stiffness-based wall stresses.

Taken together these data suggest that segmental aortic stiffnessgenerates substantial axial wall stresses that also are susceptible to ahypertensive environment.

It was also shown that segmental aortic stiffness correlates withexperimental aneurysm progression. To further investigate thesignificance of segmental aortic stiffening (SAS) as an inducer ofaneurysm growth, a temporal analysis of SAS was performed in vivo andcorrelated to aneurysm growth in the PPE model. As shown in FIGS. 6C and6D, a continuous increase in SAS after aneurysm-induction was found,peaking at d7, which was due to increasing stiffness of the PPE-treatedsegment (5-fold higher than adjacent aorta). Of note, the SAS peakcoincided with the onset of aneurysm expansion. Moreover, as shown inFIG. 6E, the magnitude of SAS at d7 correlated with subsequent aorticenlargement between d7 and d14.

As shown in FIGS. 6C and 6D, after d7, SAS declined as a result ofprogressive stiffening of the adjacent aortic segments, which wasaccompanied by decelerating aortic diameter enlargement, as best shownin FIG. 6B. As evidenced by FIG. 6C, saline-infused controls did notexhibit significantly enhanced SAS at any point during the entireobservation period.

Pro-fibrotic mechanisms accompany stiffening of AAA-adjacent segments,thereby reducing segmental aortic stiffness.

Having detected decreased SAS at d14 due to stiffening in theAAA-adjacent aorta, the underlying molecular mechanisms wereinvestigated. FIGS. 8A-8D examine the stiffening mechanisms ofAAA-adjacent aorta. FIG. 8A depicts temporal analysis of the Col1a1 andCol3a1 gene expression in the AAA-adjacent aorta compared to the AAA(PPE-treated) segment. FIG. 8B depicts temporal analysis of miR-29bexpression in the AAA-adjacent aorta compared to the AAA (PPE-treated)segment. FIG. 8C depicts in situ hybridization for miR-29b (purple-bluedye) and red nuclear counterstain in the AAA-adjacent aortic segments(original magnification 400×, scale bar 50 μm). FIG. 8D depictsrepresentative images of the aortic wall taken from AAA-adjacent aorticsegments 7 days or 14 days after PPE-treatment stained with PicrosiriusRed (upper panels; red: collagen; yellow: muscle) and Elastin VVGstaining (lower panels) (original magnification 400×, scale bar 50 μm).In these figures, n=5 for each time point, and p values denotedifferences between expression levels by Kruskal-Wallis test with Dunn'sposttest.

As shown in FIG. 8D, medial collagen deposition—a known determinant ofarterial stiffness—was remarkably enhanced in AAA-adjacent segments atd14 after aneurysm induction (compared to d7). Further, as shown in FIG.8A, expression of the collagen genes Col1a1 and Col3a1 was increased inthe adjacent segments compared to the AAA segment itself at d7,preceding the histological alterations. In line with this observation,as shown in FIG. 8B, miR29b—previously shown to be an epigeneticnegative regulator of collagen expression in AAA—was similarlydownregulated at d7. More specifically, in situ hybridization (ISH)indicated marked miR-29b downregulation within the aortic media, asshown in FIG. 8C.

In contrast to the marked pro-fibrotic changes, as shown in FIG. 8D,elastin architecture appeared unaffected in the AAA-adjacent aorta.

It was also shown that interventional reduction of segmental stiffnessreduces wall stress and aneurysm progression. To investigate thepotential causative role of segmental aortic stiffening as a mechanismdriving AAA development, the adjacent aorta next to the PPE-treatedsegment was focally stiffened by peri-aortic application of BioGlue, asurgical adhesive with a relatively high material stiffness, as shown inFIGS. 14A and 14B. More specifically, FIG. 14A depicts the abdominalaorta before glue application, while FIG. 14B depicts the aorta afterglue application to the segments adjacent to the PPE-treated segment.Please note that the scale bar in the figures represents 1 mm. As shownin FIG. 9A, glue application induced rapid and sustained stiffening ofthe adjacent aortic segments, resulting in near-equalization ofstiffness between the PPE-treated segment and the glue-treated adjacentsegments. This was reflected in a significant reduction of SAS comparedto sham-glue treated controls, as shown in FIG. 9B.

FIGS. 9A-9G depict the effects of glue-treatment on segmental aorticstiffness and aneurysm progression. FIG. 9A depicts temporal analysis ofthe circumferential cyclic strain of the glue-treated adjacent aorta(bold line) in relation to the PPE-treated segment (thin line). FIG. 9Bdepicts temporal analysis of segmental aortic stiffness (“SAS”) inglue-treated aortas compared to sham-glue-treated conditions. FIG. 9Cdepicts diameter development of the PPE-treated segment in glue-treatedvs. sham-glue-treated conditions. FIG. 9D depicts temporal developmentof SAS following delayed glue or sham treatment 7 days after PPE surgery(see arrow). FIG. 9E depicts temporal development of aortic diameterfollowing delayed glue or sham treatment 7 days after PPE surgery (seearrow). FIG. 9F depicts representative Elastin VVG staining of theaortic wall taken from native abdominal aortas (control) or PPE-treatedsegments (d14) after additional treatment of the adjacent aorta withglue or sham-glue (original magnification 400×; scale bars 50 μm). FIG.9G depicts representative Picrosirius Red staining of the aortic walltaken from native abdominal aortas (control) or PPE-treated segments(d14) after additional treatment of the adjacent aorta with glue orsham-glue (original magnification 400×; scale bars 50 μm). Please notethat EVG staining was used to depict the integrity of the medial elastinlamellae. Picrosirius Red staining aided the visualization of the aorticwall architecture and collagen remodeling. In these figures, n=7 foreach time point; p values denote differences between aortic segments inFIG. 9A and treatment groups in FIGS. 9B-9E by permutation F-test.

To exclude the possibility that aortic constriction due to segmentalglue treatment might lead to alterations of the downstream aortic flowand fluid shear stress, thereby affecting aneurysm formation, the aorticdiameter of the glue-treated segment and the downstream flow profilewere monitored. Neither luminal narrowing (data not shown) nor elevatedflow shear stress levels were detected, as shown in FIG. 15, whichdepicts shear stress occurring in the PPE-treated segment via ultrasoundmeasurements for shear stress calculation that were taken at day 7 afterPPE-induction. Further, as shown in FIG. 16, glue treatment of theadjacent aorta did not cause perturbations of its elastin architecturenor an enhanced fibrotic response, suggesting that direct mechanicalinteraction with the aortic wall caused the stiffening effect. Morespecifically, FIG. 16 depicts elastin architecture (Verhoef Van Giesonstaining, shown in the upper panels) and collagen deposition(Picrosirius Red staining, shown in the lower panels) of theAAA-adjacent aorta (day 14) with glue treatment as shown in the rightpanels) and without glue treatment as shown in the left panels. As shownin the figure, while the elastin architecture of the aortic wall isunaffected by surrounding glue treatment, collagen deposition is reduced(possibly reflecting an effect of mechanical offloading of theglue-stiffened aorta).

Further, as shown in FIG. 7C, our finite element model demonstrated thatstiffness equalization between all segments (i.e., reduction of SAS)resulted in decreased and homogenized axial stress. As discussed above,FIG. 7C depicts a segmentally stiff aorta that is initially depicted onthe left when it has not been subjected to external stiffening and alsois depicted on the right after it has been subjected to externalstiffening of the adjacent compliant segments (simulating gluetreatment) to demonstrate axial stress reduction and homogenizationinduced by the intervention.

Finally, comparing aortic diameter between glue-treated and shamglue-treated animals, with reference to FIG. 9C, it was found thatPPE-induced aortic expansion was significantly reduced when adjacentsegments were immobilized by glue application. The figure shows that theexpected rapid diameter increase between d7 and d14 was suppressed bythe glue treatment.

To further test the efficiency of delayed glue treatment on aneurysmprogression, additional experiments were performed with glueintervention at d7 post PPE, when there already is a small dilationcombined with a high segmental stiffness, as shown in FIGS. 9D and 9E.As a result, it was found that delayed glue-stiffening of theAAA-adjacent aorta also significantly reduces SAS and thereby repressesthe consecutive aneurysmal diameter progression compared to sham-gluetreated animals.

The results also show that a reduction of segmental stiffness modulatescritical features of AAA pathobiology. Since AAA formation isaccompanied by extensive extracellular matrix (ECM) remodeling,histologic analyses of the aneurysm wall was performed, focusing onelastin and collagen architecture. As shown in FIG. 9F, extensivedestruction of elastin fibers—a hallmark of aneurysm pathology—waspresent in sham-glue-treated mice on d14 after PPE infusion. Further, itis shown in FIG. 9G that Picrosirius Red staining revealed disturbedwall architecture with general wall thickening, loss of layeredstructure, and diffuse collagen enrichment. In contrast, elastinstructure and wall layering were better preserved in the glue-treatedgroup while collagen accumulation appeared less prominent.

AAA pathology includes enhanced reactive oxygen species (ROS)generation, vascular inflammation, vascular smooth muscle cell (VSMC)apoptosis and enhanced MMP activity. To assess the impact ofSAS-modulations on these endpoints, the PPE-treated aorta was analyzedat d7, which marks the peak of segmental stiffening but precedes theprominent diameter increase between d7 and d14.

In situ dihydroethidium (DHE) fluorescence was performed to monitor ROSgeneration. FIGS. 10A-10J depict the effects of glue-induced aorticstiffening on ROS generation and parameters of inflammation, apoptosisand ECM remodeling. More specifically, FIG. 10A depicts in situ DHEstaining of native abdominal aortas (control) or PPE-treated segmentsafter additional treatment of the adjacent aorta with glue or sham-glue(d7). ROS production was indicated by red fluorescence. Autofluorescencefrom elastic lamellae (depicted in green in the upper row) wassubtracted (bottom row). In this figure, the original magnification was×400 and the scale bar represents 50 μm. FIG. 10B depicts quantificationof average fluorescence from 3 high power fields of 3 different aortasper group. FIG. 100 depicts representative co-staining of macrophages(red F4/80 marker) and the green labeled cytokine IL-6 in nativeabdominal aortas (control) or PPE-infused segments (d7) after additionaltreatment of the adjacent aorta with glue or sham-glue (originalmagnification 400×, scale bar 50 μm). FIG. 10D depicts representativeco-staining of macrophages (red F4/80 marker) and the green labeledcytokine IL-1β in native abdominal aortas (control) or PPE-infusedsegments (d7) after additional treatment of the adjacent aorta with glueor sham-glue (original magnification 400×, scale bar 50 μm). FIG. 10Edepicts representative co-staining of macrophages (red F4/80 marker) andthe green labeled cytokine Ccl2 in native abdominal aortas (control) orPPE-infused segments (d7) after additional treatment of the adjacentaorta with glue or sham-glue (original magnification 400×, scale bar 50μm). Colocalization results in orange/yellow color. Nuclei are Hoechststained (blue). FIG. 10F depicts corresponding immunostaining ofactivated caspase-3 (red). FIG. 10G depicts expression of II6, Ccl2, andII1b in the PPE-infused segment (d7) after additional glue or sham-gluetreatment of the adjacent aorta, quantified in whole tissue. FIG. 10Hdepicts expression of II6, Ccl2, and II1b in the PPE-infused segment(d7) after additional glue or sham-glue treatment of the adjacent aorta,quantified in laser-captured macrophages. FIG. 10I depicts expressionanalysis of Mmp2 and Mmp9 (all vs. native control) in the PPE-infusedsegment (d7) after additional glue or sham-glue treatment of theadjacent aorta. FIG. 10J depicts expression analysis of Col1a1 andCol3a1 (all vs. native control) in the PPE-infused segment (d7) afteradditional glue or sham-glue treatment of the adjacent aorta. In thesefigures, p values denote differences between treatment groups by KruskalWallis test with Dunn's posttest (in FIG. 10B) or Mann-Whitney test (inFIGS. 10G-10J).

As shown in FIGS. 10A and 10B, PPE-treated segments exhibited enhancednuclear fluorescence compared to native controls while glue treatmentresulted in a significant decrease in ROS production.

Inflammation was quantified by aortic macrophage infiltration andcytokine analysis. As shown in FIGS. 10C through 10E, extensivemacrophage infiltration of the aortic wall was present 7 days afteraneurysm induction as assessed by immunofluorescence, accompanied byenhanced aortic gene expression of II6, Ccl2 and II1b (as best shown inFIG. 10G). Glue treatment reduced macrophage infiltration as well ascytokine expression, as shown in FIGS. 100, 10D, and 10E.

As best shown in FIG. 17, immunofluorescence additionally revealedmacrophage co-localization with each of these cytokines. FIG. 17 depictsimmunofluorescence macrophage co-staining for inflammatory cytokinesIL-6, IL-1β and Ccl2 on day 7 after PPE-treatment. Macrophages (F4/80)are stained red (left panels), while IL-6, IL-1β and Ccl2 are shown ingreen (middle panels). Merged images (right panels) demonstratemacrophages co-localize with cytokines (orange). Nuclei are stained blue(Hoechst) in merged pictures.

To further delineate the role of macrophages in vascular cytokineproduction, gene expression profiles of macrophages directly isolatedfrom the PPE-aneurysm sections were analyzed via laser capturemicrodissection (“LCM”). To this end, as shown in FIGS. 18A-18D,macrophages were micro-dissected (positive F4/80 staining) from theaortic wall and macrophage transcript enrichment was confirmed byenhanced Emr1 expression (encoding for F4/80 protein) compared torandomly captured F4/80-negative cells. Macrophages isolated fromsham-glue treatment exhibited significantly higher expression of II1b,II6 and Ccl2 compared to those from glue-stiffened samples, as bestshown in FIG. 10H.

FIGS. 18A-18D depict macrophage isolation via laser capturemicrodissection (LCM). More specifically, the macrophages (withF4/80-fluorescent label as shown in FIG. 18A) were selectively targetedas shown in FIG. 18B. Then the macrophages were isolated via LCM asshown in FIG. 18C. Macrophage transcript enrichment was confirmed viaenhanced Emr1 expression compared to LCM-isolated F4/80-negative cells.The “**” in FIG. 18D indicates p<0.001.

Assessing apoptosis, we detected enhanced capase-3 activity in theintimal and medial layer of PPE-treated aortic wall, which was reducedin the glue-treated group, as best shown in FIG. 10F.

MMP2 and MMP9 are essential for matrix macromolecule degradation in AAA.In accordance with the substantial elastin breakdown found inPPE-treated segments, both Mmp2 and Mmp9 were significantly upregulated.Glue-stabilization of the adjacent aortic segments—which preventedextensive elastin breakdown and collagen remodeling—minimized Mmpexpression, as shown in FIG. 10I. Additionally, as shown in FIG. 10J,this intervention reduced enhancement of Col1a1 and Col3a1 expressionafter aneurysm induction.

It was also shown that ex vivo segmental aortic stiffening inducesupregulation of AAA-related genes. The mechanism of SAS was examined asa driver of AAA pathogenesis by validating our in vivo findings ex vivo.More specifically, murine abdominal aortic segments were explanted andmounted onto a pressure myograph system. Aortae were then subjected tophysiologic pressure levels, cyclically alternating between 80 mmHg and120 mmHg. To simulate aortic stiffening, the “systolic” expansion ofeither the entire aortic segment (complete stiffening) or just thecentral aortic segment (segmental stiffening) was restrained by anexternally applied silicone cuff, as shown in FIGS. 11A-2, 11A-3, and13. After one hour of cyclic pressurization, aortic gene expression wasanalyzed.

FIGS. 11A-1-11D depict ex vivo aortic mechanical stimulation. Morespecifically, FIGS. 11A-1, 11A-2, and 11A-3 depict the scheme of theexperimental setup for differential mechanical stimulation of thecannulated aorta, in which cyclic strain is imposed on aunrestrained/unstiffened aorta (NoStiff) as shown in FIG. 11A-1, acompletely restrained aorta (CompStiff) as shown in FIG. 11A-2, or asegmentally restrained aorta (SegStiff) as shown in FIG. 11A-3. FIG. 11Bdepicts the gene expression of inflammation related genes II6 and Ccl2after one hour of mechanical stimulation in the 3 groups. FIG. 11Cdepicts the gene expression of matrix metalloproteinases Mmp2 and Mmp9after one hour of mechanical stimulation in the 3 groups. FIG. 11Ddepicts the gene expression of collagen genes Col1a1 and Col3a1 afterone hour of mechanical stimulation in the 3 groups. In FIGS. 11B-11D,n=5 for each condition, and p values denote differences betweentreatment groups by Kruskal-Wallis test with Dunn's posttest.

Cuffing the entire aortic segment had minimal to no effect on theexpression of inflammatory cytokines II6 and Ccl2. Segmental stiffening,in contrast, induced upregulation of these genes, as shown in FIG. 11B.Likewise, the expression of metalloproteinases (Mmp2, Mmp9) as well ascollagen genes (Col1a1, Col3a1)—quantified as indicators of activematrix remodeling—was significantly enhanced only in response tosegmental stiffening, as shown in FIGS. 11C and 11D.

The results show that the aging human abdominal aorta exhibits segmentalstiffening. In order to test whether SAS occurs naturally in the humanaorta, the aortic stiffness was assessed in three distinct locations(suprarenal, mid-infrarenal, bifurcational) along the abdominal aortasof 19 male patients ranging in age from 36 to 71 years without evidentAAA.

As shown in FIGS. 12A-12C, a significant negative correlation wasobserved between age and aortic cyclic strain in the suprarenal andmid-infrarenal as well as in the aortic bifurcation segments, suggestinggenerally enhanced stiffness in the aging abdominal aorta.

Important differences between the distinct aortic locations were alsodetected. While both the mid-infrarenal aorta and the bifurcationexhibited age-related strain reduction, the slope of strain reductionwas significantly steeper in the bifurcation segment, altering the(relative) SAS between two regions. In younger patients, the stiffnessbetween both segments was similar (SAS-1), but doubled (SAS-2) by age60, as shown in FIG. 12D. These results indicate that in addition tooverall stiffening of the abdominal aorta with age, the human abdominalaorta exhibits age-related segmental stiffening.

FIGS. 12A-12D depict segmental aortic stiffening in the aging humanabdominal aorta. More specifically, FIG. 12A depicts a correlationbetween age and circumferential cyclic strain in the supra-renal segmentof the human abdominal aorta. FIG. 12B depicts a correlation between ageand circumferential cyclic strain in the mid-infrarenal segment of thehuman abdominal aorta. FIG. 12C depicts a correlation between age andcircumferential cyclic strain in the bifurcational segment of the humanabdominal aorta. FIG. 12D depicts a correlation between age andsegmental stiffness (SAS, bifurcational segment vs. mid-infrarenalsegment) along the infrarenal abdominal aorta. In these figures, pdenotes significance level of Pearson correlation.

Discussion.

Using an established murine elastase-induced AAA model, we demonstratedthat segmental aortic stiffening (SAS) precedes aneurysm growth. Finiteelement analysis (FEA) revealed that early stiffening of theaneurysm-prone aortic segment leads to axial (longitudinal) wall stressgenerated by cyclic (systolic) tethering of adjacent, more compliantwall segments. Interventional stiffening of AAA-adjacent aortic segments(via external application of surgical adhesive) significantly reducedaneurysm growth. These changes correlated with reduced segmentalstiffness of the AAA-prone aorta (due to equalized stiffness in adjacentsegments), reduced axial wall stress, decreased production of reactiveoxygen species (ROS), attenuated elastin breakdown, and decreasedexpression of inflammatory cytokines and macrophage infiltration, aswell as attenuated apoptosis within the aortic wall. Cyclicpressurization of segmentally stiffened aortic segments ex vivoincreased the expression of genes related to inflammation andextracellular matrix (ECM) remodeling. Finally, human ultrasound studiesrevealed that aging, a significant AAA risk factor, is accompanied bysegmental infrarenal aortic stiffening.

Thus, the above example introduces the concept of segmental aorticstiffening (SAS) as an early pathomechanism generating aortic wallstress and triggering neurismal growth, thereby delineating a potentialunderlying molecular mechanisms and therapeutic targets. In addition,monitoring SAS may aid the identification of patients at risk for AAA.

AAA formation is accompanied by increased stiffness of the neurismalvessel segment compared to the normal aorta. Aneurysmal stiffeningoccurs due to profound changes in ECM organization including elastinfragmentation and enhanced adventitial collagen deposition and turnover.This example investigated aortic stiffening as a potential factordriving early AAA pathogenesis.

To explore the temporal relationship between aortic stiffening and AAAgrowth the widely-used PPE animal model was employed. As human AAAtypically occurs in the aged aorta, which exhibits progressive elastindegeneration and stiffening, the PPE model was deliberately chosen as anon-dissection type preclinical model of AAA because it not onlyphenotypically resembles many aspects of the human disease but is alsoinitiated by mild destruction of the elastin architecture (although thisis achieved enzymatically by PPE perfusion in contrast tofatigue-related elastin fracture in the human situation). Moreover,previous studies indicated that this model in particular appearssensitive to extracellular matrix/stiffness related interventions. SeeMaegdefessel L, Azuma J, Toh R, Merk D, Deng A, Chin J, Raaz U,Schoelmerich A, Raiesdana A, Leeper N, McConnell M, Dalman R, Spin J,Tsao P, “Inhibition of microRNA-29b reduces murine abdominal aorticaneurysm development,” J Clin Invest. 2012; 122:497-506.

The data in this example confirm that aortic stiffening precedesneurismal dilation. The rapid stiffening which occurred within one dayafter treatment seems to be due to early PPE-induced elastin damage, asshown in FIG. 6F. However, PPE is biologically active for no more than24 hours after perfusion. Therefore, later structural alterations of theaorta, including the pervasive elastin fragmentation observed after 14days as shown in FIG. 9F, appear to be PPE-independent.

Although the observed early and sustained stiffening of theaneurysm-prone aorta may seem counterintuitive, this finding supportsaneurysm growth as an active process, as opposed to simple passivedilation. Moreover, segmental stiffening of the abdominal aorta mayqualify as a mechanism generating wall stress.

Mechanical stress is a potent inducer of physiologic arterialremodeling. High flow-induced shear stress, elevated circumferentialstress, and increased axial stress result in increased vessel diameter,wall thickening, and arterial lengthening, respectively, to achievestress normalization. From a pathogenic point of view, mechanical forcesinduce a multitude of adverse events contributing to vascular disease,including ROS generation, apoptosis, and inflammation.

To test the hypothesis that SAS generates wall stress that precedes andtriggers early AAA growth, in silico stress-analysis employing a FEAmodel was carried out. Inclusion of a stiff segment in a more compliantaorta generates axial stress under systolic pressurization. Axial stressincreases with enhanced stiffness-gradients between stiff and non-stiffsegments, as shown in FIG. 7A. Hypertension, a known AAA-associated riskfactor, further increases axial stress in the setting of SAS, as shownin FIG. 7B. Of note, this simplified model only takes into accountstatic wall stresses, neglecting dynamic effects that may occur due tocyclic systolic-diastolic wall deformations.

In the animal model in this Example, the peak of SAS at d7 coincidedwith the onset of accelerated neurismal enlargement. Delayed AAAformation until 7 days after PPE-treatment is consistent with theinitial characterization of this model. The relationship betweenincreasing SAS and subsequent neurismal dilation was furtherstrengthened by a positive correlation between the extent of SAS at d7,and aortic diameter enlargement between d7 and d14.

To clarify the pathophysiologic significance of SAS for AAA-growth,rapid-hardening biologic glue was selectively applied to the aorticsegments adjacent to the PPE-injury site, achieving dramatic stiffeningof the adjacent aorta, detectable within one day after intervention.Subsequently, the relative segmental stiffness of the PPE-treated aortacompared to its adjacent segments (i.e, SAS) was instantly andpermanently reduced. A major finding of this study is that the(glue-induced) reduction in SAS translated into significantly reducedAAA growth. In a more therapeutic context, it was additionally foundthat delayed glue application (day 7 post PPE injury) reduced subsequentAAA progression.

To elucidate the mechanisms of this process, factors that contribute toAAA and that are moreover known to be mechanosensitive were analyzed:ROS generation, inflammation, ECM-remodeling and apoptosis. ROS levelsare locally increased in human AAA compared to the adjacentnon-aneurysmal aorta. ROS may be generated in response to mechanicalstress in endothelial cells (Ecs) as well as in vascular smooth musclecells (VSMCs), whereby mechanically activated NADPH oxidases (NOX) andthe mitochondrial electron transport chain seem to be significantsources. Mechanically generated ROS may subsequently trigger a varietyof cellular responses such as VSMC apoptosis and vascular inflammation.ROS-scavengers and NADPH-oxidase inhibition have reduced oxidativestress and aortic macrophage infiltration, and ultimately amelioratedaneurysm growth or decreased aneurysm rupture incidence in variousmurine AAA models. Decreased ROS generation was found followingglue-mediated reduction of SAS and axial stress.

AAA-formation is characterized by inflammatory remodeling of the aorticwall, and vascular inflammatory reactions are sensitive to mechanicalstress-induced signaling. For example, mechanical stress inducedpro-inflammatory mechanisms involve enhanced cytokine production viaRas/Rac1-p38-MAPK-NF-□B (leading to enhanced IL-6 expression in VSMC),as well as enhanced NF-□B-dependent expression of vascular chemokinesand adhesion molecules that facilitate monocyte adhesion to the vascularwall. Interestingly, inflammatory cells such as monocytes/macrophagesbecome mechanosensitive once attached to the vascular ECM. It has beenshown that interventional stiffening of the adjacent aorta decreasesmacrophage infiltration in the aneurysm-prone (PPE-treated) segment andreduces the aortic and macrophage-specific expression of variousinflammatory cytokines that are known to be critical for AAApathogenesis, including II1b, II6 and Ccl2.

ECM remodeling, with enzymatic breakdown of matrix macromoleculesmediated by the metalloproteinases MMP-2 and MMP-9, is another hallmarkof AAA. MMP expression is increased in human AAA, and knockout of MMP-2and MMP9 abolishes experimental AAA formation. MMP-2 and MMP-9 are alsoresponsive to mechanical stress due to cyclic stretch and enhanced flow.More importantly, axial stress induces tissue remodeling and Mmp-2activation in a model of longitudinal carotid growth. As expected, Mmp2and Mmp9 were significantly upregulated in PPE-treated aorta, as shownin FIG. 10I. Reducing SAS, and thereby cyclic axial stress, withglue-stiffening reduced expression of both MMPs.

VSMC apoptosis is another critical feature of human and experimentalAAA, and susceptible to enhanced mechanical (axial) stress. Signalingmechanisms of mechanical stress-induced VSMC apoptosis include a varietyof molecules, such as the endothelin B receptor, integrinal -rac-p38-p53signaling or Bcl-2-associated death factor (BAD). Enhanced medial layerapoptosis was identified in PPE-treated segments, which was decreased byglue-mediated axial stress reduction.

The impact of SAS on inflammation and matrix remodeling ex vivo wasfurther investigated. Segmental stiffening (induced with an externalcuff around the cyclically-pressurized aorta) resulted in significantupregulation of Mmp2 and Mmp9, Coital and Col3a1, as well as II6 andCcl2. In contrast to the in vivo situation, where enhanced bi-axialstiffness results from alterations of the inherent material propertiesof the vessel wall, the ex vivo model only simulated circumferentialstiffening by external cuffing. Due to technical limitations, thesystolic and diastolic pressure levels alternated with a frequency of3/min (normal C57BL/6 heart rate:˜450/min⁴¹). Nevertheless, the dataindicate that cyclic axial mechanical stress may directly control genesgoverning inflammation and matrix remodeling.

Stiffening of the aneurysm-adjacent aorta was observed at d14 afterPPE-induction, with subsequent reduction of aneurysm growth rate. Thismight represent an endogenous compensatory mechanism to reduce SAS andcontain AAA progression. The stiffening process was paralleled by anenhanced fibrotic response in the AAA-adjacent segments' media,including upregulated collagen expression. A previous study showed thatmicroRNA (miR)-29b is a repressor of collagen expression in AAA. SeeMaegdefessel L, Azuma J, Toh R, Merk D, Deng A, Chin J, Raaz U,Schoelmerich A, Raiesdana A, Leeper N, McConnell M, Dalman R, Spin J,Tsao P, “Inhibition of microRNA-29b reduces murine abdominal aorticaneurysm development,” J Clin Invest. 2012; 122:497-506. AnalogousmiR-29b downregulation was identified in the (VSMC-dominated) media ofthe AAA-adjacent aortic segments, consistent with miR29b-modulated VSMCcollagen production and medial fibrosis. It was previously demonstratedin the previous study mentioned immediately above that forced miR-29bdownregulation (via systemic “anti-miR” administration) is apro-fibrotic intervention reducing AAA growth. This reduction, in lightof the present example, may be partially due to acceleratedmiR-29b-dependent stiffening of the AAA-adjacent aorta.

Local aortic PPE infusion is a widely used preclinical AAA model thatexhibits many features seen in human AAA, including early disturbance ofelastin integrity. However, due to the artificial, invasive nature ofthe model, including enzymatic injury of the vessel, segmental stiffnessmight be model-specific, and not a feature of human AAA. Therefore, itwas further examined whether the human abdominal aorta exhibitssegmental stiffness that would be a contributing factor for AAAformation. Performing ultrasound-based strain analyses in three distinctlocations along the abdominal aorta (suprarenal, mid-infrarenal,bifurcation), age-dependent reduction of strain (increased stiffness)was detected, corresponding to previous observations (see O'Rourke M,Hashimoto J., “Mechanical factors in arterial aging: a clinicalperspective,” J Am Coll Cardiol. 2007; 50:1-13). As a novel finding,relatively more pronounced stiffening of the aortic bifurcation segmentwith age was detected as shown in FIG. 12C, translating into increasingSAS of the aortic bifurcation over time, as shown in FIG. 12D. Thisdistal part of the aorta has relatively low elastin content as comparedto the more proximal segments, a feature that might become functionallyrelevant with age-dependent loss of elastin. These data confirm andrefine previous observations of enhanced age-dependent stiffening of theabdominal aorta and might partly explain the significant influence ofage on AAA risk.

Of note, the segmental stiffness observed in the human abdominal aorta(SAS-2) was significantly smaller than the peak segmental stiffness inthe PPE-treated aorta (SAS-5). The study patients presumably exhibited“physiologic” stiffness segmentation that will most likely not result inAAA formation. However, segmental stiffening may have more dramaticeffects in individuals with genetic predilection for aneurysm formation.

In conclusion, the present example introduces the novel concept ofsegmental aortic stiffening as a pathogenetic factor contributing toAAA. It is proposed that degenerative stiffening of the aneurysm-proneaortic wall leads to axial stress, generated by cyclic tethering ofadjacent, more compliant wall segments. Axial stress then induces andaugments processes necessary for AAA growth such as inflammation andvascular wall remodeling, as shown in FIG. 19. More specifically, FIG.19 depicts a proposed mechanism of early AAA formation driven byage-related segmental aortic stiffening. Degenerative segmentalstiffening of the abdominal aorta induces axial stress in the stiffsegment, thereby promoting active inflammatory wall remodeling resultingin AAA formation.

From a therapeutic perspective, this example shows that mechanicallystiffening the AAA-adjacent aorta can provide a “stress shield” to limitAAA remodeling and expansion. While it could be postulated thatprotective interventional stiffening of an AAA-adjacent segment maycreate a distal stiffness gradient along the arterial tree thatpotentially triggers distal aneurysm formation, no evidence of this wasobserved during the 28-day time course of the instant model. This mayindicate that in addition to stiffness gradients other predisposingco-factors (e.g., a structurally impaired vessel matrix) may be requiredto trigger AAA formation de novo. Further, increased blood pressurelevels were not detected after interventional stiffening of theabdominal aorta that could potentially point towards negativehemodynamic side effects (See Table S1 below).

TABLE S1 Blood Pressure Measurements in Glue- or Sham- Treated Mice 7Days after PPE-Induction Glue (n = 5) Sham (n = 5) p value SBP 128 ± 2127 ± 3 0.627 DBP 100 ± 3 105 ± 3 0.826 MAP 110 ± 3 113 ± 3 0.888 PP  27± 2  25 ± 2 0.948 SBP indicates systolic blood pressure, DBP diastolicblood pressure, MAP mean arterial pressure, PP pulse pressure (= SBP −DBP).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in theirentireties.

It is to be understood that this invention is not limited to particularformulations or process parameters as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

Although the present invention has been described with reference topreferred embodiments, persons skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

1. (canceled)
 2. A method of treating a subject for a vascular aneurysmin an artery, the method comprising increasing a mechanical stiffness ofat least one vascular segment adjacent to the vascular aneurysm in thesubject.
 3. The method of claim 2, wherein the increasing the mechanicalstiffness comprises applying a surgical adhesive locally to the at leastone vascular segment.
 4. The method of claim 2, wherein the increasingthe mechanical stiffness comprises implanting an intravascular stentthat stiffens the at least one vascular segment.
 5. The method of claim2, wherein growth of the vascular aneurysm is reduced compared to in theabsence of treating the subject.
 6. The method of claim 2, wherein thesubject shows decreased inflammation in the artery compared to in theabsence of treating the subject.
 7. The method of claim 2, wherein thesubject shows decreased apoptosis in the artery compared to in theabsence of treating the subject.
 8. The method of claim 2, wherein thesubject shows decreased production of reactive oxygen species in theartery compared to in the absence of treating the subject.
 9. The methodof claim 2, wherein the subject has an early stage vascular aneurysm.10. The method of claim 2, wherein the at least one vascular segmentcomprises a vascular segment upstream of the vascular aneurysm or avascular segment downstream of the vascular aneurysm.
 11. The method ofclaim 2, wherein the at least one vascular segment comprises a vascularsegment upstream of the vascular aneurysm and a vascular segmentdownstream of the vascular aneurysm.
 12. A method of minimizing growthof a vascular aneurysm in an artery of a subject, the method comprisingselectively increasing a mechanical stiffness of at least one vascularsegment adjacent to the vascular aneurysm such that the vascularaneurysm is not directly treated.
 13. The method of claim 12, whereinselectively increasing the mechanical stiffness of the at least onevascular segment comprises applying a surgical adhesive locally to theat least one vascular segment.
 14. The method of claim 12, whereingrowth of the vascular aneurysm is reduced compared to in the absence ofselectively increasing the mechanical stiffness of the at least onevascular segment.
 15. The method of claim 12, wherein the subject showsdecreased inflammation in the artery compared to in the absence ofselectively increasing the mechanical stiffness of the at least onevascular segment.
 16. The method of claim 12, wherein the subject showsdecreased apoptosis in the artery compared to in the absence ofselectively increasing the mechanical stiffness of the at least onevascular segment.
 17. The method of claim 12, wherein the subject showsdecreased production of reactive oxygen species in the artery comparedto in the absence of selectively increasing the mechanical stiffness ofthe at least one vascular segment.
 18. The method of claim 12, whereinthe subject has an early stage vascular aneurysm.
 19. A method oftreating an vascular aneurysm, the method comprising increasing themechanical stiffness of at least one aneurysm-adjacent vascular segmentby positioning a stiffening device or stiffening composition at the atleast one aneurysm-adjacent aortic segment and not at the abdominalaortic aneurysm.
 20. The method of claim 19, wherein the stiffeningcomposition comprises a surgical adhesive, wherein the positioning thestiffening composition further comprises applying the surgical adhesiveto an outer surface of the aneurysm-adjacent vascular segment.
 21. Themethod of claim 19, wherein the stiffening device comprises anintravascular stent, wherein the positioning the stiffening devicefurther comprises deploying the intravascular stent into a lumen of theaneurysm-adjacent vascular segment.